In recent years, demand for bioethanol is rapidly increasing in other countries because of countermeasures against global warming or the need of an alternate for fossil resource. A yeast (Saccharomyces cerevisiae) having high fermentation efficiency is the leader in bioethanol production. In the '70s, studies were actively conducted concerning Zymomonas mobilis, which is an ethanol-producing bacterium. From the late 80s, ethanol production by genetic recombinant Escherichia coli was reported. S. cerevisiae or Z. mobilis, known as a bacterium producing ethanol at a high concentration is unable to use xylose or arabinose, which is a pentose. In contrast, E. coli can use all saccharides listed herein, but its productivity per individual microorganism is lower than that of S. cerevisiae or Z. mobilis. For establishment of effective ethanol-producing technology from wood-based biomass, development of a microorganism that effectively produces ethanol from pentose is an important research project. In particular, it is desired to develop a microorganism highly efficiently converting xylose, which is richly contained in a saccharified solution of wood-based biomass, to ethanol. Regarding provision of xylose fermentability to yeast, the group of Dr. Ho of Purdue University and a group from Lund University have succeeded (see FIG. 8). Meanwhile, Dr. Ingram of the University of Florida has succeeded in such provision of xylose fermentability by introducing 2 types of ethanol synthase gene from Z. mobilis. A group of the National Renewable Energy Laboratory (NREL) has succeeded in provision by introducing 4 types of xylose metabolic enzyme gene from Escherichia coli (see FIG. 8). Furthermore, xylose fermentability has been successfully provided to novel microorganisms such as Zymobacter (Zymobacter palmae) by Dr. Yanase of Tottori University and to coryneform-group bacteria by the Dr. Yukawa of the Research Institute of Innovative Technology for the Earth (RITE). However, there still remain many challenges for practical application (such as improvement in xylose fermentability (ethanol yield and fermentation rate)) of all of these genetic recombinant microorganisms.
Pichia stipitis or the like is known as a yeast capable of fermenting xylose, but its ethanol resistance is low and its xylose metabolic system is often suppressed in the presence of saccharides such as glucose. For the production of ethanol from xylose, breeding is underway by introducing genes encoding P. stipitis-derived xylose reductase (hereinafter, referred to as “XR”) and xylitol dehydrogenase into S. cerevisiae, so that the yeast acquires the ability to metabolize xylose (Non-patent documents 1, 2, and 3) (see FIG. 1).
However, such a genetic recombinant yeast is unsatisfactory, since the efficiency of anaerobic fermentation of ethanol from xylose is sill low. Moreover, the yeast is also problematic in that in the course of fermentation, an intermediate metabolite, xylitol, is accumulated, so as to lower carbon conversion efficiency. These defects are major hurdles for increasing the efficiency of continuous and/or serial fermentation processes in effective production of ethanol from wood-based biomass.
One major cause of such low ethanol conversion efficiency is an unbalanced intracellular redox status due to a difference in coenzyme dependency between xylose-metabolizing enzymes (XR and XDH) (Non-patent documents 4 and 5). Specifically, XR converts xylose to xylitol using mainly NADPH as a coenzyme for conversion to NADP+, while XDH converts xylitol to xylulose using mainly NAD+ as a coenzyme for conversion to NADH upon conversion (see FIG. 1). As described above, because of the resulting lack of balance of requirements for coenzymes between the two enzymes, the quantitative balance in coenzyme supply is disturbed. As a result, it is inferred that xylitol to xylulose conversion proceeds inefficiently, and ultimately the efficiency of xylose to ethanol conversion is lowered.
Furthermore, the fact that the activity of xylulokinase (hereinafter, referred to as XK), which is originally retained by S. cerevisiae is weak is also suggested as a cause of such low ethanol conversion efficiency (Non-patent documents 6 and 7). A method has been reported as a measure for improving the matter, which involves causing overexpression of S. cerevisiae-derived XK in addition to XR and XDH and then improving xylose to ethanol production efficiency using the recombinant yeast (see Patent document 1). It has also been reported that in such a case, expression of XR, XDH, and XK within yeast at appropriate levels is extremely important. For example, the proportions of the optimum expression levels of XR and XDH necessary to increase xylose to ethanol production efficiency are almost completely understood (Non-patent documents 8, 9, and 10). However, the optimum level of XK is controversial. Specifically, Dr. Ho of Purdue University has reported that high XK activity is important. Actually, high yields of ethanol have been obtained from xylose using the Saccharomyces yeast 424A (LNH-ST) strain, which has the ability to metabolize xylose provided via gene recombination (Non-patent document 8). In the meantime, Lund university and other groups have reported that genetic recombinant yeast strains were prepared from experimental strains (in which genes encoding XR, XDH, and XK, respectively, had been separately integrated onto different chromosomes using auxotrophic expression cassettes) and the genes are preferably appropriately expressed constitutively since excessive XK activity inhibits the growth of yeast (Non-patent documents 7 and 11).
Also, a method has been reported that involves preparing XDH (modified-type XDH) by converting its specificity for a coenzyme from NAD+ requirement to NADP+ requirement, preparing a genetic recombinant yeast co-expressing the modified-type XDH together with XR, and then producing ethanol from xylose using the genetic recombinant yeast (see Patent document 2 and FIG. 1).
In recent years, the use of ethanol or the like obtained by fermentation of a biomass resource as liquid fuel or a chemical raw material has been examined and is attracting attention. The technological development for practical use thereof is being accelerated. Therefore, economy for practical use of a biomass resource requires a yeast strain more highly capable of producing ethanol than the above yeast.    [Patent document 1] JP Patent Publication (Kohyo) No. 2000-509988 A    [Patent document 2] JP Patent Publication (Kokai) No. 2006-6213 A    [Patent document 3] JP Patent Publication (Kokai) No. 62-65679 A (1987)    [Non-patent document 1] Chu B C et al., Biotechnology Advances, Vol. 25, pp. 425-441 (2007)    [Non-patent document 2] Jeffries T W, Current opinion in Biotechnology, Vol. 17, pp. 1-7 (2006)    [Non-patent document 3] Jeffries T W et al., Applied Microbiology and Biotechnology, Vol. 63, pp. 495-509 (2004)    [Non-patent document 4] Bruinenberg P M et al., Applied Microbiology and Biotechnology, Vol. 18, pp. 287-292 (1983)    [Non-patent document 5] Koetter P et al., Applied Microbiology and Biotechnology, Vol. 38, pp. 776-783 (2004)    [Non-patent document 6] Deng X X et al., Applied Biochemistry and Biotechnology, Vol. 24/25, pp. 193-199 (1990)    [Non-patent document 7] Johansson B et al., Applied and Environmental Microbiology, Vol. 67, pp. 4249-4255 (2001)    [Non-patent document 8] Eliasson A et al., Enzyme and Microbial Technology, Vol. 29, pp. 288-297 (2001)    [Non-patent document 9] Jeppsson M et al., FEMS Yeast Research, Vol. 3, pp. 167-175 (2003)    [Non-patent document 10] Walfridsson M et al., Applied Microbiology and Biotechnology, Vol. 48, pp. 218-224 (1997)    [Non-patent document 11] Sedlak M et al., Applied Biochemistry and Biotechnology, Vol. 113-116, pp. 403-416 (2004)    [Non-patent document 12] Jin Y-S et al., Applied Microbiology and Biotechnology, Vol. 69, pp. 495-503 (2003)